Zfp281 Inhibits the Pluripotent-to-Totipotent State Transition in Mouse Embryonic Stem Cells

The cell-fate transition between pluripotent and totipotent states determines embryonic development and the first cell-lineage segregation. However, limited by the scarcity of totipotent embryos, regulators on this transition remain largely elusive. A novel model to study the transition has been recently established, named the 2-cell-like (2C-like) model. The 2C-like cells are rare totipotent-like cells in the mouse embryonic stem cell (mESC) culture. Pluripotent mESCs can spontaneously transit into and out of the 2C-like state. We previously dissected the transcriptional roadmap of the transition. In this study, we revealed that Zfp281 is a novel regulator for the pluripotent-to-totipotent transition in mESCs. Zfp281 is a transcriptional factor involved in the cell-fate transition. Our study shows that Zfp281 represses transcripts upregulated during the 2C-like transition via Tet1 and consequentially inhibits mESCs from transiting into the 2C-like state. Interestingly, we found that the inhibitory effect of Zfp281 on the 2C-like transition leads to an impaired 2C-like-transition ability in primed-state mESCs. Altogether, our study reveals a novel mediator for the pluripotent-to-totipotent state transition in mESCs and provides insights into the dynamic transcriptional control of the transition.


INTRODUCTION
Totipotency refers to the ability of a cell to generate all cell types (Lu and Zhang, 2015). In mouse embryos, zygotes and 2-cell embryos are considered totipotent cells. When the embryo develops beyond zygotes and 2-cell (2C) stages, embryos progressively lose totipotency, go through the first lineage segregation, and establish pluripotent inner cell mass (ICM) at the blastocyst stage (Lu and Zhang, 2015). An impairment in the totipotent-to-pluripotent state transition in mouse embryos leads to defects in embryonic development, indicating that the transition is crucial for embryonic development (Percharde et al., 2018;Guo et al., 2019;Tian et al., 2020). However, limited by the material scarcity, mechanistic studies of the cell-fate transition between pluripotency and totipotency are largely impeded.
Mouse embryonic stem cells (mESCs) derived from the ICM were established as a model for pluripotency study (Li and Izpisua Belmonte, 2018). The mESC culture can be maintained in the ground-naïve, the metastable-naïve, or the primed state (Li and Izpisua Belmonte, 2018). When mESCs are cultured in the metastable-naïve condition, less than 1% of ESCs spontaneous transit into a totipotent-like state. This state, named as 2-cell-like (2C-like) state, exhibits several features of 2C-stage embryos, including totipotent-like developmental potential and the expression of 2Cspecific transcripts, such as Zscan4d and MERVL repeats (Macfarlan et al., 2012;Fu et al., 2019).
Our previous studies generated an inducible 2C-like transition model, a reporter mESC cell line containing a MERVL-promoter-driven reporter, and a doxycyclineinducible Dux transgene (synDux) (Fu et al., 2019). The synDux can drive the pluripotent-to-2C-like transition in mESCs, and the reporter can indicate whether the cells are in the 2C-like state. Importantly, synDux-induced 2C-like transition recapitulates the spontaneous 2C-like transition in the mESC culture (Fu et al., 2019;Fu et al., 2020a). Thus, this cell line is a valuable tool to monitor the entry and exit of the 2C-like transition. Using this model, we constructed the comprehensive roadmap for the 2C-like transition and revealed the regulatory network controlling the transition via a genome-wide CRISPR-Cas9-mediated screen (Fu et al., 2019;Fu et al., 2020a).
By examining the screen result, we identified that Zfp281 is a candidate factor affecting 2C-like transition. Zfp281 is a transcription factor modulating cell-fate transitions through transcriptional regulation. For instance, Zfp281 inhibits the expression of naïve-pluripotent-related genes via the interaction of Tet1 and consequentially promotes the naïve-to-prime transition in mESCs (Fidalgo et al., 2016). In addition, Zfp281 inhibits the transition from pluripotent cells to extraembryonic endoderm stem cells (XENs) by interacting with polycomb repressive complex 2 (PRC2) (Huang et al., 2021). Furthermore, Zfp281 promotes mESCs to transit into trophoblast stem cells (TSCs) via recruiting COMPASS (Complex Proteins Associated with Set1) to activate TSC-related genes (Ishiuchi et al., 2019). Taken together, these results suggest that Zfp281 plays a critical role in the cell-fate transition in mESCs. To this end, we set out to examine the function of Zfp281 in the 2C-like transition.

FACS
Flow cytometry analysis was performed using the BD FACSAria Fusion SORP. Data and images were analyzed and generated using FlowJo (V10) software. The gating strategy was shown in FACS figures.

CRISPR-Cas9
The gene knockdown by CRISPR-Cas9 was performed in our previous reports (Fu et al., 2019). The sgRNA sequences are listed in Supplementary Table S2. Lentivirus was produced using the psPAX2-PMD2. G system in 293T cells. The mESCs were infected with lentivirus for 48 h in a medium containing1 μg/ ml Polybrene. After 2 days of infection, cells were cultured in a medium containing 1 μg/ml puromycin for another 8 days to select for infected cells.

Pseudo-Genome Preparation
As repeat elements tend to have multiple highly similar copies along the genome, it is relatively complex to accurately align them and estimate their expression. Hence, we created a repeat pseudo-genome. We used a slightly modified version of the RepEnrich (v0.1) (Criscione et al., 2014) software. Briefly, for each repetitive element subfamily, a pseudo-chromosome was created by concatenating all genomic instances of that subfamily along with their flanking genomics 15bp sequences and a 200bp spacer sequence (a sequence of Ns). The pseudo-genome was then indexed using STAR (v.2.5.2b) (Dobin et al., 2013), and the corresponding gtf and refFlat files were created using custom scripts and by considering each pseudo-chromosome as one gene.

Sequencing Alignment for Coding Genes
Raw reads were first trimmed using Trimmomatic (v.0.36). Illumina sequence adaptors were removed, the leading and tailing low-quality base pairs (fewer than 3) were trimmed, and a 4-bp sliding window was used to scan the reads and trim when the window mean quality dropped below 15. Only reads having at least 50-bp were kept. The resulting reads were mapped to the mm10 genome using STAR (Dobin et al., 2013) (v.2.5.2b) with the following parameters: outSAMtype BAM SortedByCoordinate-outSAMunmapped Within-outFilterType BySJout -outSAMattributes NH HI AS NM MD -outFilterMultimapNmax 20 -outFilterMismatchNmax 999 -quantMode TranscriptomeSAM GeneCounts. The generated gene expression count files generated by STAR were then used for estimating gene expression.

Sequencing Alignment for Repeats
Multi-mapped reads and reads mapping to intronic or intergenic regions were extracted and then mapped to the repeat pseudo-genome. First, the TagReadWithGeneExon command of the dropseq tools (v1.13) (Macosko et al., 2015) was used to tag the reads into utr, coding, intergenic and intronic reads using the bam tag "XF". Multi-mapped reads, intergenic and intronic reads were extracted and mapped to the repeat pseudo-genome using STAR. The STAR read counts were used as an estimate of repeat expression.

Differential Gene Expression Analysis of Bulk RNA-Seq Data
The R/Bioconductor edgeR package (v3.24.0) (McCarthy et al., 2012) was used to detect the differentially expressed genes between the different samples using the generalized linear model-based method. Genes showing more than twofold expression change and an FDR<0.0001 were considered as differentially expressed.

Functional Enrichment Analysis
Clusterprofiler was used to perform GO function enrichment and KEGG pathway annotation (Krämer et al., 2014). The associated GO and pathway enrichment plots were generated using the ggplot2 package (v3.1.0).

The Transcription Factor Zfp281 Inhibits the Pluripotent-to-2C-Like Transition
We previously performed a genome-wide screen to search for factors mediating the 2C-like transition (Fu et al., 2019). Zfp281 is one of the top candidate factors inhibiting the 2C-like transition ( Figure 1A). To validate the screen result, we used the inducible 2C-like transition model (Supplementary Figure S1A) to examine the role of Zfp281 on the 2C-like state. We design two independent sgRNA targeting Zfp281 and verify their efficiency ( Figure 1B). Our results show that Zfp281 perturbation significantly increases the population of 2C-like cells and the expression of 2C-like-state marker genes after 24-h synDux induction ( Figures 1C,D). Importantly, Zfp281 perturbation also increases the spontaneous 2C-like transition, and the expression of synDux is not altered upon Zfp281 perturbation (Supplementary Figures S1B,C), indicating that Zfp281 does not mediate 2C-like transition through synDux. In addition, Zfp281 does not bind to MERVL nor affect the expression of the MERVL-promoter-driven reporter (Supplementary Figures  S1D,E), suggesting that Zfp281-perturbation does not directly regulate the reporter in our cell model. Lastly, Zfp281knockdown by two independent siRNA significantly increases the expression of Zscan4d (2C-like-state marker gene) upon synDux induction (Supplementary Figure S1F) (Macfarlan et al., 2012). Altogether, these results suggest that Zfp281 regulates the 2C-like transition.
Zfp281 may affect the initiation, the entry, or the exit of the 2C-like transition. The initiation of the 2C-like transition is induced by Dux activation. We find that Zfp281-perturbation does not affect synDux or Dux expression (Supplementary Figures S1C,G), suggesting that the initiation of the transition is not affected by Zfp281. In addition, without initiating the 2Clike transition, Zfp281-perturbation exhibits minimal changes in the transcripts upregulated or downregulated during the 2C-like transition of metastable-naïve state mESCs (these transcripts are named as 2C-upregulated/downregulated transcripts, respectively in the following text, Supplementary Figure S1H  Table S3). This result further suggests that Zfp281 does not affect the initiation of 2C-like transition. In addition, our results show that Zfp281-perturbed 2C-like cells exit from the 2C-like state at a similar rate compared to that of control cells (Supplementary Figure S1I), implying that Zfp281 does not modulate the exit of 2C-like transition. Notably, Zfp281perturbation exhibits no effect on the cell growth within 48-h upon synDux induction, suggesting that Zfp281 does not mediate the transition through cell proliferation (Supplementary Figure  S1J). Taken together, these results suggest that Zfp281 impedes the entry of 2C-like transition. Recently, novel totipotent reprogramming method, such as RNA-splicing inhibition, has been developed for mESCs. Our data shows that Zfp281perturbation facilitates the activation of Zscan4d induced by the RNA-splicing inhibition (Supplementary Figure S1K), suggesting that Zfp281 is a general inhibitor for the pluripotent-to-totipotent state transition in mESCs.
To identify how Zfp281 shapes the transcriptome of 2C-like transition, we compare the published ChIP-seq results of Zfp281 with the RNA-seq data (Fidalgo et al., 2016). We find that although Zfp281 can bind to both 2C-downregulated and 2C-upregulated genes (Supplementary Figure S2D), the transcriptional changes of Zfp281-bound-2C-upregulated transcripts are more significant than that of Zfp281-bound-2C-downregulated transcripts ( Figure 2D).
Additionally, the change of total 2C-upregulated genes is significantly higher than that of total 2C-downregulated genes upon Zfp281 perturbation ( Figure 2E). These results suggest that Zfp281 mainly regulates 2C-like transition by affecting 2Cupregulated genes. Previously, we identified that Myc majorly affects 2C-downregulated genes during the 2C-like transition. The transcriptional regulation of Zfp281 on 2C-like transition is different from that of Myc, and we find that knockdown of Myc can indeed further increases the transcriptional changes of 2C-like-state marker transcripts in Zfp281-perturbed cells (Supplementary Figure S2E). Interestingly, we find that Zfp281-unbound 2C-upregulated genes are significantly increased upon Zfp281 perturbation, implying that Zfp281 can shape the transcriptome of 2C-like transition both directly and indirectly (Supplementary Figure S2F).
Firstly, we focus on the methylation of H3K4. The knockdown of Cxxc1 or Kmt2d significantly decreases H3K4me3 in mESCs (Supplementary Figure S3B). However, these manipulations do not consistently affect the expression of Zscan4d and MERVL upon Dux activation (Supplementary Figure S3C). These results indicate that the methylation of H3K4 does not affect 2Cupregulated genes during the 2C-like transition.
We next focus on Tet1. The major role of Tet1 is DNA demethylation (Wu et al., 2011). Interestingly, Tet1 plays a dual role in shaping the transcriptome of mESCs (Wu et al., 2011). It can activate and inhibit gene transcription by interacting with distinct epigenetic factors. Tet1 directly interacts with Zfp281 in mESCs (Fidalgo et al., 2016). In addition, the Tet family participates in the regulation of the 2C-like transition (Lu et al., 2014;Qiu et al., 2020;Huang et al., 2021). All these suggest that Tet1 contributes to the inhibitory effect of Zfp281 on 2C-upregulated transcripts during the 2C-like transition. To validate the hypothesis, we analyze the Tet1 and Zfp281 ChIP-seq data in mESCs. The majority of Zfp281-bound 2C- Frontiers in Cell and Developmental Biology | www.frontiersin.org May 2022 | Volume 10 | Article 879428 6 upregulated genes are bound by Tet1 ( Figure 3A). Furthermore, within Zfp281-bound 2C-upregulated genes, the ones that are bound by Tet1 exhibit higher transcriptional changes than those not bound by Tet1 upon Zfp281 perturbation (Supplementary Figures S3D,E), suggesting that Tet1 mediates the inhibitory effect of Zfp281 on the 2C-upregulated genes.
Afterward, we test the transcriptional effects of Tet1 knockdown in Zfp281-perturbed and control mESCs (Supplementary Figure  S3F). After Dux induction, Tet1 deficiency significantly increases the expression of 2C-like-state marker transcripts (Zscan4d, Zfp352, and MERVL) while showing no effect on these transcripts in Zfp281perturbed cells (Figures 3B-D). These results suggest that Tet1 mediates the suppression effect of Zfp281 on 2C-upregulated genes during the 2C-like transition. Notably, Tet1 knockdown exhibits a larger effect than Zfp281-perturbation on Zscan4d ( Figure 3D), indicating that Tet1 may inhibit the expression of Zscan4d via additional mechanisms other than Zfp281 (Lu et al., 2014). The diverse inhibitory effect of Tet1 on Zscan4d may contribute to the distinct genetic epistasis results of Zscan4d compared to that of MERVL and Zfp352 (Figures 3B-D).
Lastly, we find that Tet1 knockdown significantly facilitates the 2C-like transition in control cells but does not affect the 2C-like transition in Zfp281-perturbed cells ( Figure 3E). This result not only suggests that Zfp281 inhibits the 2C-like transition via Tet1 but also supports that Zfp281 mediates the 2C-like transition through transcriptional regulation.

Zfp281 Contributes to the Impaired 2C-Like-Transition Ability in the Primed-State mESCs
The mESC can be cultured in three major pluripotent states, which are the ground-naïve state (cultured with MEK inhibitor, GSK3β inhibitors, and LIF, hereafter "2iL"), metastable-naïve state (cultured with serum and LIF, hereafter "SL"), and primed state (cultured with fibroblast growth factor 2 and Activin A, hereafter "FA") (Fidalgo et al., 2016;Li and Izpisua Belmonte, 2018). The mESCs in the ground-naïve state exhibit significantly lower 2C-like transition compared to mESCs in the metastable-naive state (Macfarlan et al., 2012). One of the reasons is that ground-naïve mESC  (Fu et al., 2019). (E) The relative percentage of 2C-like cells in mESCs upon indicated manipulation. (B-E) The siTet1-1/2 represents two independent siRNA targeting Tet1, siGFP is the negative control. Dox represents doxycycline. The X in sgX refers to the gene that sgRNA targets to; sgSv40 is negative control. Shown are mean ± s.d, n = 3. p Values were calculated by the parametric one-way analysis of variance (ANOVA) test, ns = no significance, ** <0.01, *** <0.001, **** <0.0001.
Frontiers in Cell and Developmental Biology | www.frontiersin.org May 2022 | Volume 10 | Article 879428 Frontiers in Cell and Developmental Biology | www.frontiersin.org May 2022 | Volume 10 | Article 879428 8 exhibits a higher expression of Nanog, which inhibits the 2C-like transition . The 2C-like transition in primed pluripotency has not been investigated. Interestingly, primed-state mESCs show higher Zfp281 expression compared to that of naïvestate mESCs (Fidalgo et al., 2016). Given that Zfp281 inhibits the 2Clike transition, we hypothesized that primed-state mESCs might exhibit decreased potential for the 2C-like transition.
To validate the hypothesis, we firstly compare the 2C-like transition ability of mESCs cultured in ground-naïve, metastablenaïve, and primed states. Consistent with previous results, groundnaïve state mESCs exhibit impaired 2C-like transition ability than mESCs cultured in the metastable-naïve state. Interestingly, primedstate mESCs exhibit an even lower ability of 2C-like transition compared to that of ground-naïve state mESCs ( Figure 4A).
To test whether Zfp281 contributes to the decreased 2C-like transition in primed-state mESCs, we compare the effect of Zfp281perturbation on 2C-like transition in mESCs maintained in different states. Although Zfp281 deficiency increases the 2C-like cells population in each pluripotent state ( Figure 4A), the effects are distinct. The impact of Zfp281 on the 2C-like transition is most significant in primed-state mESCs ( Figure 4B). The transcriptomic analysis also suggests that Zfp281-perturbation has a more substantial effect on 2C-upregulated transcripts in primed-state mESCs than in naïve-state mESCs ( Figure 4C). On the contrary, Zfp281 exhibits a marginal impact on the 2C-like transition in ground-naïve-state mESCs ( Figures 4A,B), which is consistent with the low expression of Zfp281 in ground-naïve state mESCs. These results indicate that Zfp281 contributes to the decreased 2C-like transition in primed-state mESCs but does not play a major role in the reduced 2C-like transition in ground-naïve-state mESCs.

DISCUSSION
The cell-fate transition between pluripotent and totipotent states is crucial for embryonic development. However, it is challenging to examine the transition due to the limited relevant biological materials. The 2C-like transition in mESCs has recently become a novel model to study the transition (Fu et al., 2020b;Iturbide and Torres-Padilla, 2020). In this study, by using an inducible 2C-like transition model, we revealed that Zfp281 impedes the pluripotent-to-2C-like transition ( Figure 4D). Mechanisticwise, we showed that Zfp281 inhibits the activation of 2Cupregulated genes through the interaction with Tet1.
Previous results indicate that Zfp281 plays an important role in mediating cell-fate-transition, including the transition between naïve and primed state pluripotency. Here, we revealed a novel function of Zfp281 in mediating pluripotent-to-2C-like state transition, further supporting that Zfp281 is a master regulator for cell-identity determination.
The 2C-like transition is initiated by the transcription factor Dux and is reversible. Our results indicate that Zfp281 specifically impedes the entry of 2C-like transition but exhibits no effect on the initiation or the exit of 2C-like transition. Notably, we found that Zfp281 shows no impact on the maintenance of the 2C-like state, indicating that Zfp281 is not required for the self-renewal of the 2Clike state.
Notably, Zfp281 does not inhibit the 2C-like transition by modulating the transcription of Dux or synDux (Supplementary Figures S1C,G). Instead, Zfp281 impedes the transition by inhibiting 2C-upregulated transcripts (Figure 2). Upon Dux or synDux induction, 2C-upregulated transcripts are transcriptionally activated, and the inhibitory effect of Zfp281 on these transcripts will become more prominent. Thus, Dux or synDux can amplify the inhibitory effect of Zfp281 on the 2C-like transition.
It has been reported that the Tet family inhibits the 2C-like transition by maintaining the expression of pluripotent genes, but the individual effect of the Tet family member on the 2C-like transition has not been carefully examined (Qiu et al., 2020). In this study, we showed that Tet1 interacts with Zfp281 and inhibits the 2C-like transition through impeding the activation of 2C-upregulated genes, indicating that the Tet family plays multi-dimensional roles in the pluripotent-to-totipotent transition. Notably, although it has been reported that Zfp281 directly interacts with Tet1 (Fidalgo et al., 2016), it is theoretically plausible that Tet1 and Zfp281 suppress 2C-like transition via redundant parallel pathways.
Lastly, our study compared the potential for the 2C-like transition in ground-naïve, metastable-naïve, and primed state pluripotent stem cells. Our results revealed that primed-state mESCs exhibit decreased potential for 2C-like transition, and Zfp281 contributes to the decrease. On the contrary, Zfp281 plays a minimal role in the 2C-like transition in ground-naïvestate mESCs, suggesting the role of Zfp281 on the 2C-like transition is dependent on the pluripotent state.
In conclusion, our study reveals the function of Zfp281 on the 2C-like transition and the underlying mechanisms. It is interesting to investigate whether Zfp281 plays a similar role in totipotent mouse embryos and human ESCs.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material. The RNA-seq dataset generated during this study has been deposited to NCBI Gene Expression Omnibus (GEO, GSE201478).

AUTHOR CONTRIBUTIONS
XF contributed to the study conception and design. Material preparation, data collection, and analysis were performed by XF, XW, ZL, and HW. The manuscript was written by XF, XW, LC, and ZL. All authors commented on previous versions of the manuscript, read, and approved the final manuscript.

FUNDING
This study is supported by the National Key Research and Development Program of China (2021YFC2700101) and the hundred talents program of Zhejiang University.